The present invention is directed to the technical field of laboratory mixing and stirring devices, particularly devices that are used for dissolving or mixing materials and reagents. It is readily appreciated by those of ordinary skill in the art that thorough mixing is critical in performing processes such as chemical reactions, biological reactions and assays.
The discovery of new beneficial therapeutic agents requires the testing of chemical and biological candidates to determine whether a single candidate or class of candidates have sufficient desired characteristics to warrant further investigation and development. High throughput screening is a valuable tool for the rapid testing of large numbers of chemical and biological agents using robotics, data processing, control software, liquid handling devices and detectors. One essential, although somewhat unassuming, piece of equipment in this process is the microtiter plate, also commonly referred to as a microplate or microwell plate. A microplate typically has 6, 24, 96, 384 or even 1536 sample wells arranged in a 2 by 3 ratio rectangular matrix, such as the 8×12, 96 well microplate. Microplates having 3456 and 9600 wells have also been investigated for feasibility. The dimensions of a typical microplate are generally 5 inch by 3⅜ inch (128 mm by 86 mm) with a height of ⅜ inch to ⅝ inch (9.5 mm to 16 mm). Regardless of the number of wells, all of the wells are located in an area that is 4¼ inch by 2⅞ in (108 mm×73 mm). Therefore increasing the number of wells per plate results in increasing the density of the wells for each plate because the size of the plate is not increased. As expected, this also results in wells having smaller volumes, and thus making it difficult to mix the contents of the wells.
Microplates are flat plates having multiple wells arranged in standardized formats, in which each well serves as a test tube. Microplates are used in high-throughput (HTP) assays for such purposes as compound screening for drug discovery, diagnostic testing and genomic analyses. Microplates commonly used for HTP assays include 96-well, 384-well and 1536-well plates. The nominal capacities of the wells in these plates are 380 μl, 120 μl and 12 μl, respectively; the recommended working assay volumes are 200 μl, 80 μl, and 8 μl, respectively (see Table 1). At such small volumes, adequate mixing is difficult because of the tendency of the liquids to adhere to the wall and not to freely move. Thorough mixing is necessary to obtain reliable assay data. Mixing is critical in assays that use particulate components in test mixtures, such as bio-conjugate beads (for example polymer beads conjugated with the assay target or reporter molecules) and, sub-cellular particles, as those components precipitate without mixing. Mixing is also critical in assays using cells which grow attached to the well surfaces and do not move about in the assay medium.
TABLE 1Microplates commonly used in high-throughput assays.Recom-WellWellmendedWellDiam-WellCapac-Assayarrange-eterDepthityVolumeMicroplatement(mm)(mm)(μl)(μl)96wells8 rows of71038020012 wells384wells16 rows of3.8101208024 wells1536wells32 rows of1.7512848 wells
High throughput screening provides many benefits, one of which is the relatively small amounts of materials required. This provides the user the ability to acquire data about a large number of candidates at relatively low cost. Hence, there is a continual need to develop assay and screening processes that improves the efficiency of currently configured well plates, as well as developing processes for employing microplates having even larger numbers of wells.
However the use of currently available microplates and microplates that are in development having even greater number of wells is hampered by physical constraints resulting from the smaller wells and the corresponding smaller volumes that they can accommodate. Materials and reagents used for screening assays are often difficult to dissolve. Failure to dissolve the materials for an assay can result in inaccurate or inconsistent data. It has been shown that mixing the contents of the well can alleviate this problem (Hancock, Michael K., Medina, Myleen N., Smith, Brendan M., and Orth, Anthony P., “Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts”, Journal of Biomolecular Screening 12(1); 2007, 140-144, www.sbsonline.com).
This problem is not easily addressed because of the size of the wells and the corresponding smaller volume of materials. The smaller well size and amounts of materials make it difficult to impart sufficient agitation for thorough mixing of the contents in the well. This challenge is only exacerbated by the trend to employ higher density microplates that is microplates having a greater number of smaller wells, such as the aforementioned 384 well and 1536 well microplates.
There have been attempts to address this problem by mechanically shaking and agitating the entire microplate. However as noted previously, the size of the wells do not lend to the process of agitating the contents of the wells, and is likely to be even less effective with higher density microplates, because of the correspondingly smaller wells.
The need for adequate mixing in microplates has long been recognized and several types of mixing devices have been developed. These include orbital shakers designed for microplate mixing, magnetic stirrer systems, sonicators and acoustic mixers. Each type has its own advantages and disadvantages (Comley, John, “Microplate Mixing—Bioassay Panacea or Proven Distraction?)”
Orbital shakers for microplates have small orbiting radii (1 to 2 mm) and operate at high speeds. Efficient mixing requires shaking speeds as high as several thousand revolutions per minute. Such high-speed shaking tends to cause foaming or splashing of sample liquids, which must be avoided. In a system designed for improved mixing, stationary pins are immersed in the sample wells while the microplate is shaken on an orbital shaker at speeds as fast as several thousand revolutions per minute.
Assays using cultured cells, or cell-based assays, are increasingly used in drug discovery. Mammalian cells widely used in such assays are sensitive to mechanical stress, and shaking of microplates may cause the cells to be disrupted or dislodged from the well surfaces on which they grow (Song O. R., Kim T H, Perrodon X, Lee C, Jeon H K, Seghiri Z, Kwon H J, Cechetto J, Christophe T (2010). Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high content/throughput screening. J Biomol Screen. 15(2):138-147). Orbital shakers are unsuitable for mammalian cell-based assays.
Mixing by a magnetic stirrer requires a stirrer element placed in each well and, in general, the stirrer element must be removed from the well for sample measurement. These processes are cumbersome and require specifically designed equipment. The system is not readily adaptable to small volumes. The stirrer element may also mechanically disrupt assay components by direct contacts, such as cells growing on the well surfaces.
In a modified system, U-shaped pins equipped with a propeller-like magnetic stirrer element are immersed in the wells. The stirrer elements are then made to spin in a propeller-like motion by the use of a magnetic stirrer. This system is suitable for large volumes but is not readily adaptable to small volumes. Spinning of the stirrer element may also cause splashing of sample liquids.
Sonicators are often used for tissue homogenization and DNA shearing. It is also effective in helping dissolve materials. However, sonication cannot be used for mixing in some assays such as those using cells, sub-cellular particles or bio-conjugate beads. Similarly, acoustic mixing applied at the energy level necessary for efficient mixing is disruptive to cells or other materials. Acoustic mixing may also cause splashing of sample liquids.
Methods or devices that enable efficient yet non-disruptive and controlled mixing are an unmet need for high throughput screening. The present disclosure provides embodiments that address this unsolved need, as well as other related problems.